Team:Georgia State/Notebook/may


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Kristin/Lydia, Group C

Week 1: 5/13 to 5/17
We transformed E. coli with an overnight culture of pGAPZαC that we had previous plated from last year’s glycerol stocks. After letting the transformed cells recover overnight in the 37°C shaking incubator, we preformed minipreps on the samples and performed restriction digest with either EcoRI or Bgl II to confirm insertion of the plasmid. After confirmation, we made glycerol stocks of E. coli with pGAPZαC.
We also made an overnight culture of P. Pastoris from glycerol stocks, plated the culture, and placed the plates with growth in the refrigerator.

Week 2: 5/20 to 5/24
Our -20°C freezer, where all our enzymes were stored, was left open overnight. We conformed that our enzymes were still working by performing digestions on pGAPZαC miniprep samples and running the digestions of a 1% agarose gel at 70V for 120 minutes.
We made eight hour cultures of E. coli that we had transformed with pGAPZαA + RFP during last year’s project and stored as glycerol stocks in the -80°C freezer. We performed minipreps on the cultures then digested the them with Bgl II. We confirmed the digestion of the RFP by running it on a 1% agarose gel at 80 V for 90 minutes.
We performed midipreps on overnight cultures of the E. coli with pGAPZαC that where transformed in the previous week.
We digested pGAPZαB and pGAPZαC with EcoRI and Xba I and precipitated the DNA. We confirmed the presents of DNA in our samples by running the samples on a 1% agarose gel at 80 V for 90 minutes.
We made overnight cultures from the P. pastoris plates we had made in the previous week and used the culture to create competent P. pastoris cells that we stored as glycerol stocks in the -80°C freezer.

Week 3: 5/27 to 5/31
We digested 2µl of pGAPZαB MCS linker and pGAPZαC MCS linker that we had designed to be used to standardize pGAPZαB and pGAPZαC respectively.
We digested three samples of the pGAPZαA+ RFP miniprep from Week 2 and ran the samples on a gel at 70 V for 90 minutes.
We digested pGAPZαB and pGAPZαC minipreps with EcoRI and XbaI, performed a DNA precipitation and resuspended the DNA in 50µl of TE buffer. We ran 10µl of each sample on a gel at 70 V for 90 minutes.
We excised the DNA of pGAPZαB MCS linker, pGAPZαC MCS linker, pGAPZαB, and pGAPZαC but was unable to recover any DNA from the pGAPZαA + RFP digestions.
After finishing the gel extractions of the digested pGAPZαB and pGAPZαC, we ligated the samples of pGAPZαB and pGAPZαC with the proper MCS linker piece. Next, we transformed E. coli with either the pGAPZαB + MCS linker or pGAPZαC + MCS linker and plated the transformations on LB plates with Zeocin. The next day the plates did not show any growth so we made new LB + Zeocin plates and repeated the ligations, digestions, and transformations. On Friday, our plates still did not show growth. We made more plates, this time using low salt LB as is proper when using Zeocin. We also check the concentrations of DNA in the digested pGAPZαB + MCS Linker and pGAPZαC + MCS Linker by gel electrophoresis.